(Malvaceae) in the Mascarenes: Old Taxa on Young Islands?

DIVERSIFICATION OF DOMBEYOIDEAE (MALVACEAE) IN THE MASCARENES: OLD TAXA ON YOUNG ISLANDS?

Timothée Le Péchon,1,*,† Qiang Dai,* Li-Bing Zhang,*,‡ Xin-Fen Gao,* and Hervé Sauquet§

*Chengdu Institute of Biology, Chinese Academy of Sciences, PO Box 416, Chengdu, Sichuan 610041, China; †Université de La Réunion, Laboratoire d’Informatique et Mathématiques–Institut de Recherche en Mathématiques et Informatique Appliquées, 2 Rue Joseph Wetzell, Bat. 2, F-97490 Sainte-Clotilde, La Réunion, France; ‡Missouri Botanical Garden, PO Box 299, St. Louis, Missouri 63166-0299, USA; and §Laboratoire Ecologie, Systématique, Evolution, Université Paris-Sud, Centre National de la Recherche Scientiﬁque, Unité Mixte de Recherche 8079, 91405 Orsay, France

Editor: Félix Forest

Premise of research. The patterns of diversification of Mascarene taxa remain largely unknown in comparison to other insular systems. Traditional interpretations of insular radiations often assume that endemic taxa radiated after the origin of the insular habitats on which they were established. The Dombeyoideae (Malvaceae) sublineage endemic to Mauritius and Réunion in the Mascarenes is an ideal model to test for the signature of insular diversification. Methodology. We combined molecular sequences for a dense sample of Mascarene dombeyoids together with African, Malagasy, and Asian outgroup species. We estimated divergence times based on two calibration schemes (including or excluding geological calibration). Comparative phylogenetic methods were used to study the diversification rates and the evolution of the floral disparity in the Mascarene clade. Pivotal results. Excluding geological constraints resulted in drastically older age estimates than when we included such calibrations. Diversification patterns suggest a decrease of diversification rates through time. The low morphological disparity indicates an early partitioning of floral characters. Conclusions. The lineage diversification and the morphological disparity are consistent with traditional scenarios of insular radiation. However, the Mascarene clade is older than Réunion and Mauritius, suggesting the onset of radiation before the formation of the archipelago. The diversification might have been driven by geographical opportunity rather than ecological opportunity.

Keywords: Dombeya, island radiation, lineage diversiﬁcation, morphological disparity, the Mascarenes, Trochetia.

Online enhancements: appendix ﬁgures and tables.

Introduction the activity of the Madagascar and Indian Ocean islands biodiversity hot spot. The largest island, Réunion, possesses an Due to their similar properties (e.g., small size, isolation from active volcano and a high mountainous formation, with its the continental landmass, and distinct boundaries), oceanic is- highest peak at 3070 m. Although older and lower than Ré- lands and similar isolated habitats (such as lakes or caves) are union, Mauritius also presents a high diversity of isolated considered attractive environments for studying patterns and habitats because of the natural erosion and recent lava flows processes driving species diversification (Carlquist 1974; Giv- that have deeply modified the island (McDougall and Cha- nish 1997; Emerson 2002). The Mascarene Archipelago con- malaun 1969). As a consequence, both islands show dra- sists of three young oceanic islands (Réunion [2–5 Ma], matic topographical contrasts, and this heterogeneity has led Mauritius [8–10 Ma], and Rodrigues [8–10 Ma]; McDougall to the formation of numerous and diverse habitats (Thébaud et al. 1965; McDougall and Chamalaun 1969; Sheth et al. et al. 2009). As in many other oceanic archipelagos, the 2003) that (with the exception of Rodrigues) originated from Mascarene biota is characterized by high levels of endemism and a relatively low number of species compared with similar habitats in mainland regions (Thébaud et al. 2009). Despite 1 Author for correspondence; current address: School of Life Sci- showing interesting features for the study of evolutionary ences, University of KwaZulu-Natal, Private Bag X01, Scottsville, processes, the biogeography and patterns of diversification of Pietermaritzburg 3209, South Africa; e-mail: [email protected]. Mascarene taxa remain largely unknown in comparison to Manuscript received July 2014; revised manuscript received September 2014; other insular systems such as Hawaii or the Galapagos (Parent electronically published January 21, 2015. et al. 2008; Baldwin and Wagner 2010). Although the number

211 212 INTERNATIONAL JOURNAL OF PLANT SCIENCES of available phylogenetic studies of Mascarene taxa has in- Subfamily Dombeyoideae (Malvaceae s.l., cotton family) creased over the last few years (e.g., Maurin et al. 2007; includes ca. 350 plant species distributed in the Paleotropical Micheneau et al. 2008), only a handful of these studies (e.g., region, with the highest diversity found in Madagascar and Harmon et al. 2008a; Strijk et al. 2012, 2014) have analyzed neighboring islands (ca. 250 species; Kubitzki and Bayer diversification in light of the new framework of tests and 2003). The 24 dombeyoid species present in the Mascarenes, methodological tools now available (Rabosky 2006b; Harmon of which 23 (96%) are endemic, represent emblematic com- et al. 2008b; Glor 2010; Stadler 2011). ponents of the Mascarene biotas (Cadet 1980; Thébaud et al. In such modern approaches, the estimate of a temporal phy- 2009). Two recent phylogenetic investigations showed that logenetic framework has a central place for studying diversifi- the Dombeyoideae of the Mascarenes are polyphyletic and dis- cation patterns. The most common practice is to use temporal tributed in four clades (Le Péchon et al. 2010; Skema 2012). constraints provided by fossil records as a source of node cal- However, both studies have identified one particular well- ibration for relaxed clock molecular dating analyses (Ho and supported clade (hereafter, clade A) endemic to the archipelago Phillips 2009; Sauquet et al. 2012). However, for taxa with a (Mauritius and Réunion). This clade includes 11 species be- poor fossil record, using geological calibrations as a maximum longing to three genera: Dombeya Cav. (4 spp.; fig. 1A,1E), age bound is an occasional practice in molecular dating anal- Ruizia Cav. (1 species; fig. 1B), and Trochetia DC. (6 spp.; yses, particularly in groups that have diversified on oceanic is- fig. 1C,1D,1F). Dombeya includes many more species outside lands (e.g., Austin et al. 2004). This approach has been widely this clade and is known to be paraphyletic as a whole, but criticized, mainly because of the implicit assumption that di- phylogenetic analyses have consistently shown that the re- vergence events do not predate island ages, that is, the hy- maining species branch outside this Mascarene clade (Le pothesis that the endemic clade must have diversified in situ Péchon et al. 2010, 2013a; Skema 2012). The species in clade (Emerson 2007; Forest 2009; Heads 2011). This critical as- A also show a wide range of variation in floral, anatomical, sumption is not always verified, and using hard constraints vegetative, and reproductive characters (Friedmann 1987; Hu- based on island age may strongly impact divergence time esti- meau et al. 1999; Le Péchon et al. 2009, 2011a, 2011b; Boura mates, but very few studies have tested this impact so far (e.g., et al. 2011). Therefore, clade A represents an interesting op- Mello and Schrago 2012). portunity to test for and characterize a possible insular radia-

Fig. 1 Morphological diversity of Mascarene dombeyoids (clade A, endemic to Mauritius and Réunion). A, Dombeya populnea. B, Tro- chetia blackburniana. C, Trochetia parviflora. D, Ruizia cordata. E, Dombeya ferruginea subsp. borbonica. F, Trochetia granulata. Photographs by David Caron (A, D, E, F) and Timothée Le Péchon (B, C), BACOMAR project. LE PÉCHON ET AL.—ENDEMIC RADIATION IN THE MASCARENE DOMBEYOIDEAE 213 tion using recently developed methods for studying diversifi- Phylogenetic relationships were reconstructed using maxi- cation. In this study, we reconstruct the first time-calibrated mum likelihood (ML) and Bayesian inference (BI) on the com- phylogenetic framework for Dombeyoideae using two different bined data set. Maximum likelihood analyses were performed calibration schemes to estimate the impact of calibrations on with RAxML 7.3.5 (Stamatakis 2006), with each molecular age estimates. By using geological calibrations in one of these region assigned a separate partition using the GTRGAMMA two schemes, we aim to evaluate the impact of such practice on model of sequence evolution. The best-known likelihood tree the inference of the diversification process. From the resulting was found using default search parameters and 100 search chronograms, we examine the pattern of lineage proliferation replicates. Node support was estimated using a bootstrap (BS) and morphological disparification in clade A. We then compare analysis with 1000 replicates. Bayesian inference analyses the diversification pattern of clade A with radiations of island- were conducted using MrBayes, version 3.1.2 (Ronquist and endemic taxa and discuss the possible evolutionary processes Huelsenbeck 2003), with each DNA region treated as a separate that may have driven its diversification. partition, and run on the CIPRES cluster (Miller et al. 2010). Four chains of Metropolis-coupled Markov chain Monte Carlo Material and Methods were performed for 100 million generations, sampling every 1000th generation and discarding the first 25% of sampled trees as burn-in. We checked convergence using Tracer (Ram- Taxonomic and Molecular Sampling baut and Drummond 2007). The remaining trees were then We reconstructed the phylogeny of Dombeyoideae focusing used to calculate a majority-rule consensus tree. on clade A and estimated divergence times by combining the molecular data sets from Le Péchon et al. (2010, 2013a). Our Calibration data set included 10 of the 11 species of clade A (Le Péchon et al. 2010, 2013b; Skema 2012) and 15 additional species and In this study, we experimented with combinations of three subspecies endemic to the Mascarenes. Furthermore, 22 spe- different sources of calibration information for estimating the cies from Madagascar, Africa, and Asia were added to the divergence times among the Dombeyoideae from the Mas- matrix. Following previous molecular studies (Bayer et al. carenes: (1) the fossil record, (2) secondary calibration from a 1999; Le Péchon et al. 2010; Skema 2012), we used the genera previous molecular dating analysis, and (3) geological events fi Pterospermum and Nesogordonia as outgroups. In total, our corresponding to the ages of volcanic islands. Speci cally, we sample includes 48 taxa (including subspecies and varieties) tested two different calibration schemes, including two or four fi fi fi and four DNA regions: two intergenic spacers (trnQ-rps16 constraints applied to speci c nodes (table 1; g. 2). The rst and trnD-psbM) and one intron (rpl16 intron) of chloroplast scheme used only one minimum age constraint from the fos- DNA, plus the internal transcribed spacers (ITS) of nuclear sil record and one maximum age constraint derived from a DNA. previous study (see below). The second scheme used the same calibrations plus the age of oceanic islands as two additional maximum age constraints. Each of these calibrations was as- Sequence Alignment and Phylogenetic Analysis signed a uniform age prior and is described in detail below. The DNA sequences of each region were aligned separately Stratigraphic ages were converted into absolute ages by using using BioEdit (Hall 1999). In the rpl16 intron, a particular re- the geological timescale of Gradstein et al. (2012). gion (position 438–530) of uncertain alignment was excluded to avoid any bias in the phylogenetic reconstruction. Indels Fossil calibration and molecular backbone analysis. Most were treated as missing data. The final combined data set in- of the fossil taxa assigned to the Dombeyoideae consist of fos- cluded 48 taxa and 2937 characters (voucher information in sil wood (Wheeler et al. 1994) and, less frequently, leaves app. A). We used the Akaike information criterion (AIC) as (Antal and Prasad 1996), but most of these fossils lack apo- implemented in jModelTest, version 0.1.1 (Posada 2008), to morphic characters, making their phylogenetic placement dif- select a model of substitution for each DNA region; the GTR 1 ficult. After a survey of the paleobotanical literature, we se- G model was selected in each case. lected one fossil related to our group of study.

Table 1 Summary of Calibration Points and Their Parameters for Dating Analyses Type of constraints Calibration point Source Calibration nature Calibrated node Calibration parameters Age (Ma) Root age, crown group Wang et al. 2009 Secondary calibration 1 Maximum age 88 Malvales Sphinxia ovalis Reid and Chandler 1933 Fossil 2 Minimum age 48 Maximum age estimates McDougall and Geologic 3 Maximum age 10 of Mauritius Chamalaun 1969 Maximum age estimates Gillot et al. 1994 Geologic 4 Maximum age 5 of Réunion 214 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 2 Maximum clade credibility (MCC) tree from the Bayesian relaxed clock (BEAST) analysis. Clade A is colored in blue, and taxa endemic to the Mascarenes are shown in boldface. Nodes 1–4 denote the four nodes where calibration points were applied (depending on calibration scheme). The green and yellow vertical dashed lines represent the oldest (most conservative) geological age estimates of the emergence of Mauritius and Réunion, respectively. The dark blue horizontal lines denote the 95% credibility intervals. Posterior probabilities, bootstrap support values, mean ages, and 95% highest posterior density values for all nodes are given in table B2 (available online) following node numbers in this ﬁgure. Mad p Madagascar, Mau p Maurice, Réu p Réunion, Rod p Rodrigues.

Sphinxia ovalis Reid & Chandler (hereafter, Sphinxia) has licates, with tree bisection and reconnection branch swapping been reported in the Early Eocene formations of the London and the Bayesian majority-rule consensus tree of extant taxa Clay (England, ca. 48.6–55.8 Ma; Reid and Chandler 1933). enforced as a backbone constraint. After MP searches, we ex- This fossil taxon is composed of several fruits and seeds, amined the set of alternative most parsimonious positions of which have been proposed to be related to the extant genera Sphinxia. We then determined the ﬁnal placement of the fossil Dombeya and Trochetia. For assessing the phylogenetic place- by using the most recent common ancestor of all alternative ment of Sphinxia, we used a molecular backbone approach positions. Following this method, Sphinxia was assigned to (Sauquet et al. 2009). For this analysis, we were limited to four node 2 and given a conservative minimum age of 48 Ma (ﬁg. 2; morphological characters observed in Sphinxia. A matrix of table 1). these four characters recorded for 49 taxa (i.e., 48 extant taxa and Sphinxia) was compiled based on the literature (Arènes Secondary calibration. According to Wang et al. (2009), 1959a, 1959b; Kubitzki and Bayer 2003; Le Péchon et al. the crown group age of Malvales ranges from 88 to 72 Ma. 2009, 2011b; Skema 2012) and observations of living collec- Wikström et al. (2001) obtained 67–71 Ma for this age, and tions in the Mascarenes and Madagascar (table B1; tables B1– Magallón and Castillo (2009) obtained 33.9 Ma for the di- B7 available online). A maximum parsimony (MP) analysis vergence of Bixaceae and Malvaceae. Therefore, 88 Ma can was performed using PAUP*, version 4.0b10 (Swofford 2001), be seen as a conservative maximum age for crown group through heuristic searches of 100 random taxon addition rep- Malvales. Because Dombeyoideae are well nested in the LE PÉCHON ET AL.—ENDEMIC RADIATION IN THE MASCARENE DOMBEYOIDEAE 215

Malvales (Bayer et al. 1999), crown group Dombeyoideae are et al. 2012). Each partition (DNA region) was treated with the younger than crown group Malvales. Thus, in spite of the risk optimal substitution model previously selected by jModelTest associated with secondary calibration, our use of 88 Ma as a (Posada 2008). We conducted three independent Markov maximum age for crown group Dombeyoideae here (node 1; chain Monte Carlo (MCMC) runs for 50 million generations. fig. 2; table 1) may be regarded as safe and conservative. Each MCMC was sampled every 1000th iteration, generating 50,000 chronograms. We discarded 25% of samples as burn- Geological calibration. The Mascarene archipelago is a vol- in (37,500 in total). Mixing of the chains and convergence of canic hot spot, and the three main islands have never been in the runs were checked in Tracer 1.5 (Rambaut and Drum- contact with any continental mass. The age estimates of such mond 2007). Using LogCombiner, we merged the remain- insular systems can be used as maximum age constraints for ing 112,500 trees from the two independent MCMC runs, clades endemic to them, under the assumption that such clades and the maximum clade credibility (MCC) chronogram was began to diversify on these islands (i.e., excluding a priori a constructed with TreeAnnotator (Drummond and Rambaut scenario in which they started diversifying elsewhere and then 2013). moved jointly to the island). In the Mascarenes, Mauritius has been considered to be the oldest island, and its emergence Diversification Rate Analyses was estimated to 8–10 Ma (McDougall and Chamalaun 1969). Age estimates for Réunion were often given in the range of 2–3 We used the ML and Bayesian (MCC) chronograms, ob- Ma, but the activity of the main volcano started around 5 Ma tained with r8s and BEAST, respectively, to investigate pat- (Gillot et al. 1994). The geological history of the third island, terns of lineage diversification within clade A. Analyses were Rodrigues, is more complex and controversial. Formerly con- performed using the packages Ape (Paradis et al. 2004), Laser sidered to be the youngest island of the archipelago, Rodri- (Rabosky 2006a), Geiger (Harmon et al. 2008b), and TreePar gues is now understood to be at least as old as Mauritius (Stadler 2011) in R (R Development Core Team 2008). (Shapiro et al. 2002; Thébaud et al. 2009; Strijk et al. 2014). We tested whether clade A experienced a temporal modifi- For calibrating our phylogeny, we used the upper age estimates cation in its diversification rates using the birth-death (BD) (i.e., 10 Ma for Mauritius and 5 Ma for Réunion), which re- likelihood method implemented in Laser (Rabosky 2006a, flected the oldest potential land available for colonization. We 2006b). We fit six diversification models on the ML and MCC therefore applied the safest and most conservative implemen- chronograms. To represent the uncertainty in the phylogenetic tation of these geological constraints. A maximum age con- reconstruction and dating estimates (i.e., r8s and BEAST), we straint based on the oldest age of Mauritius (10 Ma) was ap- also ran this analysis on (1) 1000 phylogenies randomly sam- plied to node 3, which corresponds to clade A (strictly endemic pled from the BEAST posterior and (2) the 1000 BS replicates to Mauritius and Réunion). In addition, a maximum age con- of the fixed topology (ML tree). Among these six models, two straint based on the oldest age of Réunion (5 Ma) was applied assume a constant rate of diversification, the pure-birth (PB) to node 4 (clade composed of species endemic to Réunion). We and birth-death models; two are modified versions of the note that node 4 is only weakly supported and that future Yule model, the Yule-2 rate and Yule-3 rate models (Rabosky analyses might reveal some non-Mascarenese species to be 2006a); and the last two are density-dependent (DD) models nested in it. However, if this were the case, our maximum age (Rabosky and Lovette 2008). We tested and selected the best constraint derived from geology applied to the most recent model by computing the DAICRC, where RC is the rate con- common ancestor of these Mascarenese species would still stant. However, the two DD models implemented in Laser apply under the assumption that the Mascarene-endemic sub- cannot accurately account for extinction (Etienne et al. 2012). clades diversified on Réunion. As a consequence, we also used a new likelihood method implemented in the TreePar package (Stadler 2011) on the MCC chronograms. This method tests for constant species diversifi- Divergence Times cation, or constant rate (CR), DD without extinction (DD 2 E), For estimating divergence times, we used two different re- and DD with extinction (DD 1 E; Etienne et al. 2012). Finally, laxed clock methods, each calibrated with the two scenarios we also used the g statistic (CR test; Pybus and Harvey 2000) above. We used r8s, version 1.71, to estimate divergence times to further test whether a temporal slowdown in diversifica- with penalized likelihood (PL; Sanderson 2002), using the best- tion rate can be detected in clade A. Although this monophyletic scoring tree obtained with RAxML (hereafter, ML tree) and group encompasses higher proportions (i.e., 83%) than the after pruning out the earliest-diverging taxon (Nesogordonia). minimum recommended 80% of species (Cusimano and Ren- The optimal smoothing parameter was determined using cross- ner 2010), we also tested the effect of missing species on the validation analysis with the additive penalty. The confidence g statistic (Monte Carlo constant rates test; Pybus and Harvey intervals for the ML analysis were obtained using 1000 boot- 2000). Procedures for performing these tests are detailed in strap resamplings on a fixed topology (ML tree), with re- “Procedure B1” in appendix B (available online). sampling carried out using RAxML (Stamatakis 2006). Di- vergence dates were estimated from the resulting 1000 trees Disparity through Time using r8s with settings as described above but also using the smoothing parameter determined for the original data set. In order to characterize the pattern of morphological diver- Bayesian estimation of divergence times was performed in sification, we measured 12 selected traits (see “Procedure B2” BEAST, version 1.7.2, using the uncorrelated lognormal in app. B.) in 481 flowers of 156 individuals, representing all (UCLN) model with a Yule prior on speciation (Drummond species included in clade A (1–46 individuals per species). The 216 INTERNATIONAL JOURNAL OF PLANT SCIENCES values were log transformed for analysis. To reduce the di- Table 2 mensionality of our data and to detect correlations among Results of Molecular Dating Analyses for Clade A, characters, we performed four different phylogenetic principal Endemic to Réunion and Mauritius component analyses (pPCA) following Revell (2009) and according to sex (male or female) and calibration scheme (with or Scheme, calibrations, method Crown age clade A (Ma) without geological age constraints). Detailed procedures are Scheme 1: given in “Procedure B2.” We used disparity through time SC, F: (DTT; Harmon et al. 2003) and the morphological disparity PL 34.6 (25.4–43.3) index (MDI) to examine the diversification of morphological UCLN 24.6 (11.8–40.8) characters within clade A. Tracing DTT onto a phylogeny re- Scheme 2 quires fully resolved phylogenetic relationships; consequently, SC, F, G: PL 10 (10.0) we used the MCC tree (fig. 2). This method compares the UCLN 8.7 (6.2–10.0) morphological disparity, estimated among and within sub- clades in relation to the global disparity at all time steps in Note. Age estimates are reported as mean age and 95% credibility p p p our topologies, to phenotypic disparity generated from a null intervals. SC secondary calibration, F fossil calibration, G p p model of 1000 simulations under a multivariate Brownian geological calibration, PL penalized likelihood, UCLN uncorrelated lognormal. motion (Harmon et al. 2003). A rapid decline of the average subclade disparity and lower MDI values than predicted under Brownian motion simulations suggests an early partitioning dependent linear) were always significantly positive (table B3). of morphological differentiation into distinct adaptive zones Therefore, these results provide evidence for variable rates and a slowdown in phenotypic diversification rates (Harmon during lineage diversification of clade A. The preferred model et al. 2003; Later et al. 2010; Later and Pennell 2013). These selected by DAIC was a density-dependent linear model, and different analyses were performed in R using Geiger (Harmon RC the difference with other variable-rate models was relatively et al. 2008b). high and significant (14.48; table B3). Analyses using TreePar showed similar trends. For both schemes of calibrations, the Results CR model was rejected against DD models. Among these models, DD 2 E was always preferred to DD 1 E with moderate ! ! Phylogenetic Analyses and Divergence Time Estimates support (2.0 DAICMCC 3.9; table B4). Furthermore, g statistic results (detailed in table B5) and the lineage-through-time Maximum likelihood (RAxML) and Bayesian (MrBayes) (LTT) plots of clade A are consistent with the DAIC results phylogenetic analyses yielded similar and congruent results. RC and provided further evidence for a slowdown in diversifica- We summarized the phylogenetic relationships in figures 2, tion rates during the history of the clade (fig. 3; table B5), re- B1, and B2 (figs. B1, B2 available online). Species of Dom- gardless of the calibration scheme and the dating method used. beyoideae from the Mascarenes are distributed in four clades For clade A, LTT plots showed an initial high rate of diversi- (fig. 2). Among them, clade A encompasses 12 taxa. This clade fication followed with a distinct decrease of this rate and slow endemic to Mauritius and Réunion is strongly supported by accumulation of lineages from this point to the present. both analyses (BS p 93, PP p 1). A second endemic clade (restricted to Réunion, node 4) was found in all of our analyses. However, this monophyletic group obtained much Evolution of Morphological Disparity p weaker support (not supported in ML; PP 0.65). For clade The four different pPCAs gave similar results; a large pro- fi A, we summarized the mean nodal ages and con dence in- portion of the dissimilarity is explained by the three first axes tervals (CIs) estimated with both analyses in table 2 (for full (88%–94% of the variance; table B6). We therefore retained details, see table B2). For both calibration schemes (i.e., with these first three axes from the pPCA for plotting the four or without geological calibrations), r8s and BEAST analyses analyses (table B6) according to sex (male or female) and cali- led to congruent results. However, PL intervals of node age bration scheme (scheme 1 or scheme 2). Visual observations of estimates are generally larger and include those found with the DTT plots (fig. 4) for clade A showed the disparity to be BEAST, and mean PL ages were older than those estimated less than expected under a Brownian motion model. These re- with BEAST. In both analyses, using internal geological con- sults were confirmed by the negative values of the MDI (greater straints (scheme 2) led to drastically younger estimates for the than 20.16; table B7). The disparity was more partitioned same node (table 2). For example, using BEAST and under among rather than within the two main lineages (i.e., Ruizia calibration scheme 1, crown age estimates for clade A vary cordata, and Dombeya spp. and Trochetia spp.), which tend between 11.8 and 40.8 Ma, whereas the age of this clade varies to occupy different regions of the morphospace. between 6.2 and 10.0 Ma when geological calibrations were applied (table 2). Discussion

Patterns of Diversiﬁcation in the Endemic Lineage Old Taxa and Young Islands: Biogeographical Implications In all of our analyses (i.e., regardless of calibration scheme The dating estimates from the two scenarios showed dra- and dating method), differences between the best rate-constant matic differences (table 2). Using only fossil constraint and model (pure-birth) and the best rate-variable model (density- secondary calibration, the age estimates of clade A appeared LE PÉCHON ET AL.—ENDEMIC RADIATION IN THE MASCARENE DOMBEYOIDEAE 217

Fig. 3 Lineage-through-time (LTT) plots for clade A, endemic to Réunion and Mauritius, according to calibration schemes and dating methods. The solid lines represent the LTT inferred from the BEAST maximum clade credibility (MCC) tree or the maximum likelihood (ML)– r8s tree. The colored areas indicate the 95% conﬁdence interval for the phylogeny generated using 1000 trees randomly sampled from the posterior distribution (BEAST analysis) or from the 1000 bootstrap resamplings on the ﬁxed topology (ML-r8s).

much older than the geological dating of Mauritius and Ré- group. A pattern of old taxa on young islands may be explained union (tables 2, B2; ﬁg. 2). According to Le Péchon et al. by the stepping-stone hypothesis. Ancestors of clade A may (2010), the diversity of the Mascarene dombeyoids resulted have colonized former nearby islands and later dispersed to from four independent events of dispersal/colonization from the Mascarenes, where we now observe the extant diversity. Madagascar to the Mascarenes. Under this scenario, the Mas- These former islands have been submerged, leading to com- carene endemic lineages would have originated after the for- plete extinction of their biota. In the Indian Ocean Region, mation of the archipelago and diversiﬁed in situ. However, the stepping-stone hypothesis is corroborated by geological this scenario, explicitly assumed by the maximum age con- evidence (Sheth et al. 2003) in combination with sea-level straints of calibration scheme 2, is inconsistent with the inde- curves and bathymetry information, which together support pendent age estimates from calibration scheme 1, suggesting the presence of several large islands near the Mascarene ar- a different interpretation of the biogeographic history of the chipelago (Thébaud et al. 2009; Warren et al. 2010). This

Fig. 4 Plots of disparity through time (DTT) inferred from the maximum clade credibility (MCC) tree for clade A, according to sex and calibration scheme. Solid lines indicate the actual disparity of subclade floral morphology. Dashed lines depict median expected disparity and shaded areas show the disparity expected for a sample of 1000 simulations under a Brownian model of evolution. ♂ p male flowers, ♀ p female flowers, ⚥ p hermaphroditic flowers. 218 INTERNATIONAL JOURNAL OF PLANT SCIENCES pattern of old taxa on young islands could thus be more wide- of the Mascarenes, during episodes of sea lowstand when sev- spread than expected. In oceanic islands, and in the Masca- eral potential large islands were available for colonization and renes, in particular, other taxa have been identified as being differentiation (i.e., geographic opportunity), and then lin- older than the islands on which they reside (e.g., Monimia eage proliferation rates may have decreased when such islands [Monimiaceae]: Renner et al. 2010; Badula [Primulaceae]: became immersed. Clade A may thus have arisen from non- Strijk et al. 2014; and Hyophorbe [Arecaceae]: Cuenca et al. adaptive speciation driven by geographic isolation and genetic 2008; Galápagos tortoises: Caccone and Gibbs 1999; and drift (Rundell and Price 2009). Hillebrandia [Begoniaceae] in Hawaii: Clement et al. 2004). Although uncertainties remain on the true ages of all of these Conclusions clades, this accumulated evidence therefore suggests that the assumption behind the use of maximum age constraints based Using a calibration scheme excluding geological constraints, on island ages might be incorrect in many cases and thus war- our crown group age estimates for clade A (endemic to Mau- rants great caution in interpreting results from studies using ritius and Réunion) are older than the accepted age for the such constraints. For this reason, the rest of our discussion emergence of the contemporary Mascarene Islands. An al- focuses on the results from calibration scheme 1. ternative biogeographical scenario is proposed to explain in- consistencies between geological dating of the islands and the molecular dating estimates. The colonization of the Radiation of Clade A: Signal for a Typical Mascarene Islands may have occurred through former islands Pattern of Insular Diversification? (now submersed) in the Indian Ocean, which may have played In birth-death likelihood analyses, the pattern of lineage the role of stepping-stones. While the biogeographical sce- proliferation in clade A shows evidence for density-dependent nario combined with results from birth-death likelihood and diversification. Under this model, the diversification rates are disparity-through-time analyses are consistent with the radia- relatively high in initial stages of the radiation and then sharply tion signature of island-endemic groups, the pattern of old taxa decline as a function of the number of species in existence. This on young islands is inconsistent with traditional scenarios of fi result is further evidenced by the significant negative values of insular diversi cation. Therefore, we propose an alternative fi the g statistic, indicating a decrease in diversification rate, and scenario in which the slowdown in diversi cation rates is the the global shapes of the LTT plots. Despite the limitations of result of limited geographic range expansion. Studies focus- current methods for detecting early burst pattern in phenotypic ing on patterns of lineage accumulation in Mascarene taxa disparification (see Later and Pennell 2013), DTT plots and remain scarce. By applying similar integrative approaches to the MDI negative values in clade A indicate that the burst of additional Mascarene clades, a comprehensive framework will lineage proliferation is paired with an early burst pattern in the eventually allow a better understanding of the evolutionary and evolution of morphological characters. biogeographic history of the Mascarene biota as a whole. The global pattern of clade A, therefore, combines different elements (i.e., biogeographical scenario, pattern in lineage, and Acknowledgments morphological diversification) suggesting that diversification of clade A is consistent with other examples of island-endemic We are grateful to the Forestry Services of Mauritius, the radiations (e.g., Darwin’s finches: Sato et al. 2001; Hawaiian National Park of Réunion, and the National Parks and Con- lobeliads: Emerson 2002; Givnish et al. 2009) but also with servation Service for collecting permission and general assis- verbal theories of adaptive radiation (Simpson 1953; Schluter tance in Mauritius and Réunion; and to the Conservatoire 2000). Indeed, an early burst pattern in morphology and lin- Botanique National de Mascarin, the Mauritian Wildlife eage accumulation could be linked to ecological opportunities Foundation, and Nature Océan Indien for field assistance in (Losos and Ricklefs 2009, 2010; Yoder et al. 2010) offered by Mauritius and Réunion. We thank Luke Harmon for discus- new environments such as lakes or recently emerged oceanic sion; Cláudia Baider, Thomas Haevermans, Jean-Noël Labat, archipelagos (e.g., the Mascarenes and other potential currently and Thierry Pailler for access to the REU, MAU, and P herbaria submerged islands in the Indian Ocean). collections; David Caron, Jean-Yves Dubuisson, Luc Gigord, However, in Hawaiian lobeliads and Darwin’s finches (and Pierre Gigord, Laurence Humeau, and Jean-Bernard Pausé many other examples), island-endemic groups have diversified for help during the BACOMAR project; Mélanie Chazalet, in situ rapidly after colonizing oceanic archipelagos (Sato et al. Jacques Fournel, Claire Cécile Juhasz, Léo Mende, Pierre Sta- 2001; Givnish et al. 2009). The clade A pattern of old taxa on menoff, and Maëva Techer for collecting morphological data; young island indicates diversification before the formation of and Joeri Strijk, Isabelle Véa, and anonymous reviewers for the Mascarenes, and this characteristic is inconsistent with helpful comments on early versions of the manuscript. This traditional scenarios of insular diversification. Other potential study is included in the BACOMAR project, supported by the processes (i.e., not necessarily linked to ecological opportunity) University of La Réunion, the Région Réunion, the European may also lead to a similar pattern in timing and tempo of clade Union, and the Ministère Français de La Recherche. Funding is A diversification (Pigot et al. 2010; Etienne and Rosindell from the Open Laboratory of Ecological Restoration and Bio- 2012; Moen and Morlon 2014). Based on simulation studies, diversity Conservation of Chengdu Institute of Biology, Chi- Pigot et al. (2010) showed that the slowdown could also be the nese Academy of Sciences (CAS; grant 2011Y1SB10); a CAS consequence of limited geographic range expansion (i.e., geo- Research Fellowship for International Young Researchers graphic opportunity) instead of saturated ecological niches. (grant 31150110463) provided support to T. Le Péchon dur- Diversification rates may have been high before the formation ing finalization of this study. LE PÉCHON ET AL.—ENDEMIC RADIATION IN THE MASCARENE DOMBEYOIDEAE 219

Appendix A

List of taxa used in this study, with origin, voucher and collection information, and GenBank number for trnQ-rps16, rpl16, psbM-trnD, and ITS (in that order). Abbreviations are as follows: Mau p Mauritius; Réu p Réunion; Rod p Rodrigues; Cult. CBNB p Culture Conservatoire Botanique National de Brest; MNHN p Muséum National d’Histoire Naturelle de Paris. Dombeya acutangula Cav., Madagascar; Labat 3654 (P), GU937945, -, GU937896, GU938038. Dombeya acutangula subsp. acutangula (Rod), Cult. CBNB; Le Péchon 131 (P), GU937947, GU937995, GU937898, GU938040. Dombeya acutangula subsp. rosea Friedmann Mau1 (Mau), Mauritius; Le Péchon 153 (P), GU937948, GU937996, GU937899, GU938041. Dombeya acutangula subsp. rosea Mau2 (Mau), Mauritius; Le Péchon 154 (P), GU937949, GU937997, GU937900, GU938042. Dombeya amaniensis Engl., Tanzania; Phillipson 4834 (P), GU937950, GU937998, GU937901, GU938043. Dombeya blattiolens Frapp. ex Cordem., Réunion; Le Péchon 294 (P), GU937951, GU937999, GU937902, GU938044. Dombeya brevistyla Arènes, Réunion; Labat 2863 (P), GU937952, GU938000, GU937903, GU938045. Dombeya burgessiae Gerr. ex Harv. and Sond, Tropical greenhouses of MNHN Paris; Specimen 15936, GU937953, GU938001, GU937904, GU938046. Dombeya cacuminum Hochr., Cult. CBNB; Le Péchon 133 (P), GU937954, GU938002, GU937905, GU938047. Dombeya ciliata Cordem., Réunion; Le Péchon 262 (P), GU937955, GU938003, GU937906, GU938048. Dombeya delislei Arènes, Réunion; Le Péchon 114 (P), GU937956, GU938004, GU937907, GU938049. Dombeya elegans Cordem. var. elegans, Réunion; Le Péchon 18 (P), GU937957, GU938005, GU937908, GU938050. Dombeya elegans var. virescens Cordem., Réunion; Le Péchon 272 (P), GU937958, GU938006, GU937909, GU938051. Dombeya farafanganica Arènes subsp. endrina, Madagascar; Razakamalala et al. 2114 (MO), GU937959, GU938007, GU937910, GU938052. Dombeya ferruginea Cav. subsp. borbonica Friedmann, Réunion; Le Péchon 22 (P), GU937960, GU938008, GU937911, GU938053. Dombeya ferruginea subsp. ferruginea, Mauritius; Le Péchon 155 (P), GU937961, GU938009, GU937912, GU938054. Dombeya ficulnea Baill., Réunion; Le Péchon 3 (P), GU937962, GU938010, GU937913, GU938055. Dombeya formosa Le Péchon & Pausé, Le Péchon 84 (P, REU), KC222207, KC222209, KC222208, KC222206. Dombeya lucida Baill., Madagascar; Ravelonarivo et al. 2011 (MO), GU937963, GU938011, GU937914, GU938056. Dombeya macrantha Baker, Madagascar; Labat 3671 (P), GU937964, GU938012, GU937915, GU938057. Dombeya mauritiana Friedmann, Mauritius; Le Péchon 129 (P), GU937965, GU938013, GU937916, GU938058. Dombeya pilosa Cordem., Réunion; Le Péchon 65 (P), GU937966, GU938014, GU937917, GU938059. Dombeya populnea Baker, Mau- ritius; Le Péchon 141 (P), GU937967, GU938015, GU937918, GU938060. Dombeya populnea, Réunion; Le Péchon 80 (P), GU937968, GU938016, GU937919, GU938061. Dombeya punctata Cav., Réunion; Le Péchon 1 (P), GU937969, GU938017, GU937920, GU938062. Dombeya reclinata Cordem., Réunion; Le Péchon 6 (P), GU937970, GU938018, GU937921, GU938063. Dombeya rodriguesiana Friedmann, Rodriguez; Le Péchon; 160 (P), GU937971, GU938019, GU937922, GU938064. Dombeya rottleroides Baill., Madagascar; SW 60080 (P), GU937972, GU938020, GU937923, GU938065. Dombeya sevathianii Le Péchon & Baider, Mauritius; Le Péchon 143 (P), GU937973, GU938021, GU937924, GU938066. Dombeya sp. 252, Madagascar; Rakotonirina et al. 252 (MO), GU937974, GU938022, GU937925, GU938067. Dombeya sp. 277, Madagascar; Rakotonirina et al. 277 (MO), GU937975, GU938023, GU937926, GU938068. Dombeya sp. 310, Madagascar; Rakotonirina et al. 310 (MO), GU937976, -, GU937927, GU938069. Dombeya superba Arènes, Madagascar; Rakotonirina et al. 293 (MO), GU937977, GU938024, GU937928, GU938070. Dombeya tiliacea Planch., South Africa; Phillipson 4834 (P), GU937978, GU938025, GU937929, GU938071. Dombeya umbellata Cav., Réunion; Le Péchon 115 (P), GU937980, GU938026, GU937931, GU938073. Dombeya viburniflora Boj., Madagascar; Labat 3771 (P), GU937981, GU938027, GU937932, GU938074. Helmiopsis bernieri (Baill.) Arènes, Madagascar; Capuron 20937 (P), GU937982, GU938028, GU937933, GU938075. Helmiopsis pseudopopulus (Baill.) Capuron, Madagascar; Capuron 27412 (P), GU937983, -, GU937934, GU938076. Nesogordonia crassipes (Baill.) Capuron, Madagascar; Rakotonirina et al. 385 (MO), GU937984, -, GU937935, GU938077. Nesogordonia suzannae Labat, Munzinger and O.Pascal, Mayotte; Berthelot 1327 (P), GU937985, GU938029, GU937936, GU938078. Pterospermum, Cult. Tropical greenhouses of MNHN Paris; Specimen 5616 (P), GU937986, GU938030, GU937937, AY083661. Ruizia cordata Cav., Réunion; Le Péchon 81 (P), GU937987, GU938031, GU937938, GU938079. Trochetia blackburniana Bojer ex Baker, Mauritius; Le Péchon 144 (P), GU937988, GU938032, GU937939, GU938080. Trochetia boutoniana Friedmann, Mauritius; Le Péchon 147 (P), GU937989, GU938033, GU937940, GU938081. Trochetia granulata Cordem., Réunion; Le Péchon 101 (P), GU937990, GU938034, GU937941, GU938082. Trochetia parviflora Boj., Mauritius; Le Péchon 162 (P), GU937991, GU938035, GU937942, GU938083. Trochetia triflora DC., Mauritius; Le Péchon 162bis (P), GU937992, GU938036, GU937943, GU938084. Trochetiopsis erythroxylon (G. Forst.) W. Marais, Cult. CBNB; Le Péchon 132 (P), GU937993, GU938037, GU937944, GU938085.

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